Growth of Pseudomonas fluorescens on iron-deficient medium did not affect the levels of several enzymes presumed to be involved in the biosynthesis of the siderophore by this organism, including, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, glutamate dehydrogenase, glutamine synthetase, and N2-acetylornithine-5-aminotransferase. The level of glutamate synthase was considerably lower in iron-deficient cells as compared to iron-sufficient cells. The concentration of glutamine synthetase in P. fluorescens was found to be 4-5 times greater than that found in Escherichia coli when grown under derepressing NH ion 4-limiting conditions. Although activity of the enzyme is regulated by an adenylylation cascade, metabolic regulation of this cascade appears to be different from that previously demonstrated in E. coli, since an unconventional pattern of adenylylation of the enzyme is observed when the organism is grown on either glycerol, succinate, malate, or fumarate. The glutamine synthetase from P. fluorescens has been purified to homogeneity and many of its properties have been determined.